In systemic autoimmune diseases, B cells secrete pathogenic autoantibodies and also present autoantigens to autoreactive T cells. These B cell functions are evidently required for pathogenesis as B cell deficient lupus- prone mice lack all signs of disease and most importantly, patients with a variety of autoimmune diseases including lupus and RA can be effectively treating by depleting B cells. How autoreactive B cells are activated in autoimmunity and how this is prevented in health are thus two very central questions. We have been intensively studying these issues and, along with many in the field, have made progress in understanding the unique features of autoreactive B cell activation and its regulation. We have been using the AM14 rheumatoid factor (RF) Ig transgenic mouse system to investigate the regulation of autoreactive B cells in normal and disease states. In the previous period, a major contribution was the finding that AM14 B cells are spontaneously activated in MRL/lpr mice to make large numbers of AFCs. This occurs mainly outside of GCs, at unique extrafollicular sites, where somatic hypermutation also occurs. This is likely a feature of anti-DNA Abs and also likely applies to other lupus-prone mice as well as in lupus patients, thus highlighting the general nature of the extrafollicular pathway in autoreactive B cell activation. Here we propose to further investigate this novel pathway of autoreactive B cell activation using the AM14 system, in particular a new version of it that has the AM14 Vh knocked in to the endogenous locus, where it can switch and mutate, We will investigate the origins of the extrafollicular RF response, taking advantage of our ability to stimulate it with IgG2a anti- chromatin in vivo, in a TLR-dependent manner. In Aim 1, we will: a) identify which cell types follicular, marginal zone, and/or B1 are capable of recruitment into the extrafollicular response; b) l test the roles of FcRs and DCs in promoting extrafollicular activation and plasmablast generation; c) determine on which cell types TLRs need to be expressed to promote RF B cell activation; and d) test the roles of BAFF/APRIL in promoting differentiation, class-switch and mutation. Autoreactive B cells have long been known to carry downstream Ig isotypes and contain somatic mutations, properties also associated with memory B cells. In Aim 2, we will test the hypothesis that autoreactive B cells generate true memory; alternatively, continuous presence of autoantigen maintains a state of constant activation. The answer to this question is fundamental to understanding chronic autoimmunity, memory and for defining optimal therapeutic approaches. In surprising contrast to autoimmune mice, we found that normal BALB/c mice produced numerous RF+ GCs, but made little detectable autoAb and AFCs. Further, the patterns of somatic mutations reflected failed positive selection, indicating a previously unrecognized tolerance checkpoint. In Aim 3, we will determine how these autoreactive GCs are regulated in normal mice, testing the roles of several key regulatory molecules and cells. Finally, we test the hypothesis that these autoreactive GCs in normal mice fail to generate memory cells.Project narrative. This work seeks to understand how autoreactive B cells, which make autoantibodies that promote systemic autoimmune diseases, are activated in disease- prone animals. It also investigates how healthy animals prevent the activation of these same B cells. Since B cells are absolutely required for these diseases, and depleting them can induce remission in humans, this work is at the crux of understanding how autoimmune diseases like lupus occur and how to better treat them.